Fused lobed anther and hooked stigma affect pollination, fertilization and fruit set in mango

Abstract

Mango malformation is the most threaten disease that limits mango production, worldwide. For a long time, due to its complex nature, the cause and causal agents were strongly disputed. Diverse Fusaria, including Fusarium mangiferae, are known to be associated with the disease. There are indications that augmented level of endogenous ethylene in response to various abiotic and biotic stresses alters the morphology of reproductive organs. Here, scanning electron microscopy (SEM) of healthy and malformed reproductive organs of mango cv. Baramasi was performed to compare the functional morphology. The SEM study revealed that anthers of hermaphrodite healthy flowers were bilobed with large number of turgid pollen grains whereas malformed flowers showed fused lobed anthers with scanty deformed pollen grains. Furthermore, the stigma of healthy flowers exhibited a broad landing pad as compared to malformed stigma which showed hooked and pointed tip. All these impaired morphology of male and female reproductive organs lead to failure of sexual reproduction. This is the first evidence to show fused lobed anther with impaired pollen grains and hooked stigma with poor stigmatic receptivity are mainly responsible for restricting the pollen germination and pollen tube growth. Here we suggest that abnormal development of anthers and pistils is due to endogenously produced stress ethylene. Further, added load of cyanide, a byproduct of ethylene biosynthesis, may also contribute to the development of necrosis which lead to desiccation of anther and pistil during hypersensitive response of plants.

Introduction

Mango (Mangifera indica L.) production is reported to be badly hit by several diseases and environmental stresses when plants are exposed to open field. Currently, malformation in mango is the most important disease of mango that received great attention not only because of its widespread and destructive nature but also because of its etiology and control which is not absolutely defined till date.¹ The disease limits mango production in several tropical and subtropical countries of the world and is responsible for 50-80 percent economic losses every year.² Malformation in mango was first reported in India in 1891.³ The disease has also been confirmed in most mango growing countries such as Pakistan, the Middle East, Egypt, South Africa, Brazil, Sudan, Central America, Mexico and USA, Cuba, Malaysia, Australia, Israel, UAE, Bangladesh and Sultanate of Oman. The disease has been variously attributed to be acarological, viral, fungal and physiological in nature.⁴ The various Fusaria, including Fusarium mangiferae, were found to be associated with the disease.⁵ However, the latest citations confirm that ethylene play a role in causing malformation.⁶ Multiple biotic or abiotic stresses give signals which usually stimulate ethylene production.⁷ Stress ethylene production due to wounding injury caused by pollen tube penetration after pollination has been reported.⁸ Desiccation of ovary and style was due to endogenously produced stress ethylene during stress.⁹ The male reproductive development is more sensitive to environmental stresses than female reproductive development which represents certain development and sterility genes.¹⁰ The scanning electron microscope (SEM) performed by Scholefield (1982) and many others has a potential to reveal ultrastructure of plant.¹¹ There are many reports where flower initiation, differentiation, development pattern and morphology were compared using SEM.^12-14 However, to my knowledge, observation on morphology of pistil and stamen of mango panicle has not been performed yet. Therefore, the objective of this study is to examine the ultrastructure of healthy and malformed reproductive organs using scanning electron microscopy.

Results

In case of male reproductive organs, the healthy anther was clearly bilobed with large number of pollen grains emerging out that are sufficient enough to ensure pollination (Fig. 1A). In contrast, malformed hermaphrodite flowers exhibited fused lobes of anther along with scanty pollen grains (Fig. 1B and C). Similarly, the case where pollen surface character of flower was taken into account, the healthy flowers exhibited turgid pollen surface with prominent exine pattern and no abnormal villi-like structures were observed (Fig. 2A and B). On the other hand, malformed flowers were appeared as a sagged with wrinkled pollens surface along with abnormal villi-like structures. This flabby and flaccid surface of pollen grains represents an abnormal morphology (Fig. 2 C–E). Morphology of malformed flower was insufficient to facilitate proper pollination

Figure 1. Scanning electron micrographs of healthy and malformed anthers of hermaphrodite flower of mango cv. Baramasi. Bilobed anther with large number of pollen grains in flower of healthy panicle, scale bar: 100 µm (A) and scale bar: 50 µm (B). Fused lobes anther with scanty pollen grains in flower of malformed panicle, scale bar: 100 µm (C).

Figure 2. Scanning electron micrographs of healthy and malformed pollen grains of hermaphrodite flower of mango cv. Baramasi. Healthy turgid pollen grains, scale bar: 5 µm (A) and scale bar: 2 µm (B). Malformed pollen, scale bar: 5 µm (C), scale bar: 2 µm (D) and scale bar: 5 µm (E).

In female reproductive organs, the healthy flower exhibited a broad landing pad on the top of style, which functions as a platform for pollen reception and facilitates stigma-pollen adhesion. Its hydration allows the pollens to germinate which leads to proper fertilization of ovary and consequently fruit set (Fig. 3A and B). In contrast, abnormal stigmatic surface was observed in stigma of malformed hermaphrodite flower. The malformed flower exhibited hooked stigma. The tip of stigma was observed to be pointed and it lacked landing pad at the top of the style, meant for receiving pollens. Due to hooked and pointed stigma, the stigma-pollen adhesion is altered, this leads to alteration of hydration and germination of pollen grains on the stigmatic surface. Thus there is failure of fertilization, hampering fruit set (Fig. 3C and D).

Figure 3. Scanning electron micrographs of healthy and malformed sigma of hermaphrodite flower of mango cv. Baramasi. Healthy stigma with a broad landing pad, scale bar: 50 µm (A) and scale bar: 50 µm (B). Malformed stigma with hooked and pointed tip, scale bar: 50 µm (C) and scale bar: 100 µm (D).

Discussion

Mango produces a large number of flowers, which in the same year results in good fruit set. Malformation causes abnormal inflorescence which affect mango production at large scale. Stamens and pistils are found to be affected by malformation disease in early stage of flowering which leads to failure of fertilization and thereby fruit set. Inadequate supply of mineral nutrients affects pollen viability, pollen germination, pollen tube extension and stigma receptivity.¹⁵ Deficiency in carbohydrate metabolism in the anther leads to abnormal pollen development in many plants¹⁶ which in turn affects the pectin substances required for the cell wall of growing pollen tube.¹⁷ There are several abiotic and biotic stresses which cause metabolic imbalance (Fig. 4). Temperature also play an important role in causing abnormal reproductive organs, so called floral malformation¹⁸ by stimulating ‘stress ethylene’ production.¹⁹ Fusarium mangiferae associated with the disease also has the capacity to produce ethylene itself which seems to add the ‘stress ethylene’ pool in mango floral tissue.⁶ As pollen tube penetration is a wounding factor, wound-induced ethylene might be one of the early events after pollination.⁸ The flower parts differed depending on the treatment used: wounding and desiccation accelerated the ethylene production in ovaries and styles; ethylene exposure resulted in enhanced ethylene emission of petals and auxin induced remarkable amount of ethylene released from leaves.²⁰ There are multiple reports which showed that scanning electron microscopy (SEM) of mango tissue have been extensively used for ultrastructural studies in mango.^21,22 However, to my knowledge, SEM for ultrastructural morphology of pistil and stamen of panicle in relation to physiological aspect of malformation has not been performed yet. Here, we find that abnormal characters of mango flower such as fused lobed anther with scanty pollen grains, sagged and wrinkled pollens surface and abnormal villi-like structures, hooked stigma and abnormal stigmatic surface etc. resembles with the characters caused by ethylene. Malformed pollen grains those are impaired in their germination and malformed stigma indicates poor stigma receptivity which restrict the pollen germination and pollen tube growth leading to fertilization failure. Moreover, the malformed egg and embryo contribute towards the impaired fertilization. Several reports support that desiccation of ovary and style in cut flowers has been observed due to endogenously produced stress ethylene during wound and water stress.⁹ The production of “stress ethylene” has been clearly identified as the degradation product of 1-aminocyclopropane-1-carboxylic acid (ACC) which is derived from methionine.²³ The methionine cycle involves at a higher pace in the production of ‘stress ethylene’, explains the higher content of cyanide which affects healthy flower tissue leading to respiratory rate.²⁴ The abnormal flowers are either not suitable for pollination or pollinated flowers do not lead to proper fertilization and complete sexual reproduction, thereby fruit set. Beta- cyanoalanine synthase is primarily responsible for cyanide detoxification in plants.^25,26 Inability of malformed floral tissue to metabolize the added load of cyanide may contribute to the development of necrosis during hypersensitive response of plants. Level of endogenous ethylene levels were induced in malformed inflorescence under influence of temperature stress.²⁷ The responses of pollen development to low and high temperature regimes have been observed. The microsporogenesis in mango is sensitive to low and high temperature stress and pollen tube kinetics and dynamics were different under temperature stress.^28,29 Future studies require to be focused on biochemical and gene expression studies associated with ecophysiological advances which may provide better clues about floral abortion.

Figure 4. The probable causes of mango malformation are ascribed as abiotic and biotic factors which lead to metabolic imbalance and stress ethylene production.

Material and Methods

Plant materials

Stamens and pistils were collected from hermaphrodite flowers of healthy and malformed panicles of sixteen years old plants of mango cv. Baramasi grown in orchard section of the Department of Plant Physiology, Horticulture Research Centre, G.B. Pant University of Agriculture and Technology.

SEM procedure

The pistils were plucked from the flowers with the help of a sterilized forceps and were dehydrated using a graded ethanol series. It was dried by keeping the samples in acetone overnight and then the stigmatic surface was observed under scanning electron microscope using an accelerating voltage of 6 kV and a spot size of 125 nm (JEOL, JSM-6610 LV). With some specimens, up to 30 min observation was possible. Similarly, the Stamens (anthers along with the pollens) were excised with the help of a sterilized forceps and it was observed under SEM at -28°C and low vacuum. All images were computer processed.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.